Post-traumatic stress disorder (PTSD) is a maladaptive and debilitating psychiatric disorder characterized by an extreme sense of fear at the time of trauma occurrence, with characteristic re- experiencing, avoidance, and hyperarousal symptoms in the months and years following the trauma. PTSD has a prevalence of approximately 6%, but can occur in up to 25% of subjects who have experienced severe psychological trauma, such as combat veterans, refugees, and assault victims. The differential risk determining those who do vs. those who do not develop PTSD is multi-determined: 1) it is in part genetic, with approximately a 30-40% risk heritability for PTSD following trauma; 2) it in par depends on sex, with women having approximately twice the risk as men to develop PTSD following trauma; and 3) it in part depends on past personal history, including adult and childhood trauma and psychological factors which may differentially mediate fear and emotion regulation. Pituitary adenylate cyclase-activating polypeptide (PACAP) has previously been identified as a critical regulator of the stress response across species. We have recently shown that PACAP and its PAC1 receptor (PAC1R) may be critical mediators of abnormal processes following psychological trauma, such as with posttraumatic stress disorder (PTSD) in humans. We recently found, in heavily traumatized human subjects, a sex-specific association of PACAP blood levels with fear physiology, PTSD diagnosis and symptoms in females (N=64, replication N=74, p<0.005). Using a tag-SNP genetic approach (44 single nucleotide polymorphisms, SNPs) spanning the PACAP (ADCYAP1) and PAC1R (ADCYAP1R1) genes, we found a sex-specific association between a single SNP (rs2267735) and fear and PTSD symptoms. The SNP residing in a putative estrogen response element (ERE) within PAC1R, predicts fear response, PTSD diagnosis and symptoms in females (combined initial and replication samples: N=1237; p<2x10-5). The presence of this SNP is inversely correlated with PAC1R mRNA expression, and methylation of PAC1R is associated with PTSD (p < 0.001). Complementing these human data, we found that PAC1R mRNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP/PAC1R pathway are involved in abnormal fear responses underlying PTSD. This proposal aims to extend these initial findings with a larger sample size, identifying peripheral markers of PACAP/PAC1 pathway activity as a biomarker for PTSD. We will further extend these data by examining the role of estrogen, PAC1R DNA methylation / histone acetylation, and PAC1R mRNA expression on PTSD symptoms and fear physiology in traumatized female subjects. We will further examine the mechanisms of PAC1 regulation using human lymphoblast cell lines of differing PAC1 rs2267735 genotypes. PUBLIC HEALTH RELEVANCE: There is a significant need for a more mechanistic understanding of the neural circuitry of fear and its inhibition. We have recently utilized convergent genetic approaches to identify the PACAP/PAC1 receptor pathway as being associated with PTSD and with dysregulation of conditioned fear in humans. We will extend these data by examining the mechanisms by which estrogen, epigenetic modulation, and genetic polymorphisms differentially modulate PAC1 gene expression in humans.